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- PDB-8j26: CryoEM structure of SARS CoV-2 RBD and Aptamer complex -

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Basic information

Entry
Database: PDB / ID: 8j26
TitleCryoEM structure of SARS CoV-2 RBD and Aptamer complex
Components
  • AM032-4
  • AM047-6
  • Fab heavy chain (REGN10987)
  • Fab light chain (REGN10987)
  • Spike protein S1
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM/DNA / Aptamer / SARS CoV-2 RBD / Inhibitor / NapdU / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM-DNA complex
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
DNA / DNA (> 10) / Spike glycoprotein
Similarity search - Component
Biological speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRahman, M.S. / Jang, S.K. / Lee, J.O.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2019M3E5D6063871 Korea, Republic Of
CitationJournal: Molecules / Year: 2023
Title: Structure-Guided Development of Bivalent Aptamers Blocking SARS-CoV-2 Infection.
Authors: Md Shafiqur Rahman / Min Jung Han / Sang Won Kim / Seong Mu Kang / Bo Ri Kim / Heesun Kim / Chang Jun Lee / Jung Eun Noh / Hanseong Kim / Jie-Oh Lee / Sung Key Jang /
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.
History
DepositionApr 14, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 12, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: AM032-4
A: Fab light chain (REGN10987)
B: Fab heavy chain (REGN10987)
C: Spike protein S1
D: AM047-6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,9876
Polymers110,7665
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules ED

#1: DNA chain AM032-4


Mass: 15288.496 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain AM047-6


Mass: 16446.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Antibody , 2 types, 2 molecules AB

#2: Antibody Fab light chain (REGN10987)


Mass: 23909.330 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
#3: Antibody Fab heavy chain (REGN10987)


Mass: 26705.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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Protein / Sugars , 2 types, 2 molecules C

#4: Protein Spike protein S1


Mass: 28415.693 Da / Num. of mol.: 1 / Fragment: RBD
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTC2
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RBD_Fab_AM032-4_AM047-6 complex / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.111 MDa / Experimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 / Strain: Wuhan
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: p42(modified pFastbac)
Buffer solutionpH: 7.5
Details: 1mM MgCl2, 0.15% amphipol A8-35 and 0.003% cymal-6 additive added during sample preparation
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
225 mMTris-Hydrogen ChlorideTris-HCl1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Aptamer AM032-4 and AM047-6 mixed with RBD_Fab complex at 1:1.2 molar ratio and incubate 1hr on ice before preparing grids
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 2.5 ul of samples placed on grid before plunge frozen with 5s blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 700 nm / Cs: 2.73 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.78 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10018
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.1.1particle selection
2EPUimage acquisition
4cryoSPARC4.1.1CTF correction
7UCSF Chimera1.16model fitting
9PHENIX1.21rc1_4895:model refinement
10Coot0.9.6 ELmodel refinement
11cryoSPARC4.1.1initial Euler assignment
12cryoSPARC4.1.1final Euler assignment
13cryoSPARC4.1.1classification
14cryoSPARC4.1.1 and 4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1938408 / Details: Particles picked using TOPAZ train
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212000 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT)
Details: Final map generated using 3D Flex reconstruction following non-uniform refinement
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-ID
17KGJ

7kgj

E7KGJE
26XDG

6xdg

A6XDGA
36XDG

6xdg

C6XDGC
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035752
ELECTRON MICROSCOPYf_angle_d1.2428215
ELECTRON MICROSCOPYf_dihedral_angle_d32.6281424
ELECTRON MICROSCOPYf_chiral_restr0.042838
ELECTRON MICROSCOPYf_plane_restr0.002721

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